1. Technical Field
The present invention relates to a method and reagents for a fluorescence polarization assay procedure for determining fetal lung maturity from a sample of amniotic fluid, and to a method for preparing the reagents.
2. Background Art
Since the discovery in the early 1970's that fetal lung maturity can be assessed from amniotic fluid collected via amniocentesis, the management of preterm deliveries has greatly improved. A variety of methods have been developed to measure some feature of amniotic fluid surfactant, each of which possesses both advantages and disadvantages. The methods currently used relate fetal lung maturity to the composition of the surfactant secreted by the fetal lungs. The most widely used test measures the lecithin/sphingomyelin (L/S) ratio of the phospholipids in the fetal lung surfactant. An L/S ratio greater than 2.0 is generally taken to indicate fetal lung maturity. This test is tedious, requiring 2 to 4 hours to perform, and the large number of individual variations in technique results in poor reproducibility between laboratories.
The foam stability index (FSI) test is another method used to assess fetal lung maturity. A sample of amniotic fluid is shaken, forming a layer of foam at its surface which is stable for several minutes. In the presence of 40-50% ethanol, the foam layer disappears rapidly. The maximum concentration of ethanol at which the foam remains is called the foam stability index. An index of greater than 0.47 is generally taken to indicate fetal lung maturity. This method has the disadvantage of requiring a large volume of amniotic fluid. In addition, the subtle difference between a "stable" and "unstable" foam layer creates difficulties in reading a result.
The use of fluorescence polarization for monitoring fetal lung maturity was first described by Shinitzky et al. in 1976 [Br J Obstet Gynaecol (1976), 83, 838]. The technique involves mixing the dye diphenyl hexatriene (DPH) with a sample of amniotic fluid. The dye associates with the liposomes formed by the surfactants present in the amniotic fluid. The sphingomyelin present in amniotic fluid from immature fetuses forms liposomes having high microviscosity. In this environment the motion of the dye is restricted and, upon excitation with plane-polarized light, a high degree of polarization is retained in the fluorescence. In the amniotic fluid from mature fetuses the surfactant liposomes contain a high level of lecithin which decreases the microviscosity. This allows the dye to have greater freedom of motion, and the fluorescence polarization is thereby decreased. A fluorescence polarization of less than 0.310 is taken to indicate fetal lung maturity. While Shinitzky's method was shown to be predictive of lung maturity, it has several disadvantages: (1) the results show a poor correlation with the more widely accepted L/S ratio; (2) the required instrumentation is expensive and frequently unavailable in hospitals; and (3) the stock solutions of reagents for the test are highly unstable and must be prepared daily.
Recent studies by Tait et al. [Clin Chem (1986) 32/2 248-254], Foerdor et al. [Clin Chem (1986) 32/2 255-259] and Ashwood et al. [Clin Chem (1986) 32/2 260-264] have made use of the Abbott TDx.sup.R fluorescence polarization analyzer to measure the fluorescence polarization in amniotic fluid of the dye 1-palmitoyl-2,6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl phosphatidyl choline (NBD-PC): ##STR1## NBD-PC is a synthetic derivative of lecithin having solubility and binding characteristics very similar to those of lung surfactant. When added to amniotic fluid, the dye should become part of the liposomes of lung surfactant, with the fluorophore associated with the hydrophobic region of the aggregate. Just as with DPH, the microviscosity of the environment of the dye in the liposome should affect its fluorescence polarization. The fluorescence polarization was seen to be high in immature samples and to decrease with increasing maturity, as with the DPH system. Good correlation was seen with both the L/S ratio and with the clinical outcome of the pregnancy.
The present invention involves the use of N-[-N-palmitoyl-N-(4-nitrobenz-2-oxa-1,3-diazole)-L-lysine]-2-aminoethanol ,N(trimethylaminoethanol)phosphate (PC16), a fluorescent probe similar inactivity to NBD-PC but with its structure modified so as to enhance its long-term chemical stability: ##STR2## The dye has been tested in amniotic fluid and in a variety of solutions developed to simulate features of amniotic fluid. Results presented herein clearly show the mechanism of its activity to involve association of the dye with the protein albumin as well as with surfactant. The fluorescence polarization or fluorescence intensity results can be calibrated and translated to a surfactant/albumin ratio, which can be reported as a highly repeatable index to fetal lung maturity.